The present invention relates to a method for racemizing N-acylamino acids using racemase, and further, a method for reacting the racemized N-acylamino acids with L-aminoacylase or D-aminoacylase to produce optically active amino acids corresponding to the respective amino acid species.
N-acylamino acid racemase (hereinafter, abbreviated to xe2x80x9cNAARxe2x80x9d) does not react with amino acids but specifically racemizes N-acylamino acids. Enzymes with NAAR activity have been found in Actinomycetes including the genera Streptomyces, Amycolatopsis, and Sebekia (Japanese Patent No. 2712331). A strain, Amycolatopsis sp. TS-1-60, which produces an acylamino acid racemase, a method for producing this enzyme (Unexamined Published Japanese Patent Application (JP-A) No. Hei 6-205668), and a DNA fragment encoding the acylamino acid racemase derived from this strain (JP-A Hei 4-365482) have been disclosed. However, only two NAARs, one of which is derived from Streptomyces sp. Y-53 and the other from Amycolatopsis sp. TS-1-60, have been isolated and purified, and their substrate specificity have been clarified.
Both of these NAARs catalyze only limited types of acylamino acids depending on their substrate specificity. The activity of NAAR derived from Streptomyces sp. Y-53, when the activity on N-acylmethionine is taken as 100, is 50 or higher on N-acylleucine, N-acylphenylalanine, and N-acylvaline, but less than 50 on N-acyltryptophan, N-acylalanine, and N-acylaspartic acid. In addition, the activity of NAAR derived from Amycolatopsis sp. TS-1-60, when the activity on N-acylmethionine is taken as 100, is 50 or higher on N-acylphenylalanine and N-acylvaline but less than 50 on N-acyltryptophan, N-acylalanine, N-acylaspartic acid, and N-acylleucine.
The present inventors previously succeeded in isolating the NAAR gene from Sebekia benihana and in expressing the recombinants thereof (Abstracts of the annual meeting of the Society for Bioscience and Bioengineering, Japan, 1999, p. 166). Although S. benihana is known to have N-acylamino acid racemase activity on N-acyl-L-methionine, the substrate specificity of the enzyme has not specifically demonstrated.
Racemization of acylamino acids is an important step for producing optically active amino acids. Enzymes have excellent catalytic functions with substrate specificity, reaction specificity, and stereospecificity. Stereospecificity of enzymes, with some exceptions, are nearly absolute.
Recent precise research has increased the importance of optically active substances for use in drugs, pesticides, feeds, and perfumes. Since optical isomers sometimes have quite different biological activities, techniques for specifically obtaining one particular isomer are important. For example, D(R)-form thalidomide has no teratogenic activity, but its L(S)-form shows strong teratogenicity. In fact, the use of thalidomide racemate caused the drug injury incidents by thalidomide. In case where one enantiomer shows an effective biological activity and the other enantiomers have no such activity, coexistence of these enantiomers may not only reduce the total activity but also inhibit the activity of the effective enantiomer competitively. As a result, the biological activity of the racemate is reduced to half or less of the activity of the effective enantiomer. Accordingly, it is industrially important to obtain (synthesize or optically resolve) optically pure enantiomers.
For this objective, a method in which racemates are synthesized and then optically resolved has been widely used. However, an unnecessary enantiomer is always produced as a by-product with the procedure of resolution after synthesis; a problem of efficiently utilizing the raw material remains unsolved. Even if the recovered by-product is reused as the raw material, a definite amount of the by-product is always produced. Therefore, enzymatic optical resolution has drawn attention because it does not produce by-products and a bulk of liquid waste. Enzymatic optical resolution is a method of specifically producing a desired enantiomer by utilizing enzyme specificity. Since unnecessary enantiomers are barely synthesized by this method, it is easy to obtain products of high optical purity. In addition, this method is also advantageous in efficiently utilizing the raw material. The racemase activity is useful for synthesizing racemates as substrates to be used either in optical resolution or in enzymatic synthesis of specific enantiomers. Thus, NAAR is required for catalyzing racemization of acylamino acids.
For example, D-tryptophan is one of important D-amino acids used as medicinal raw materials, etc. D-tryptophan can be obtained by deacylating N-acyl-DL-tryptophan. However, racemase capable of efficiently catalyzing racemization of N-acyltryptophan to N-acyl-DL-tryptophan is not yet known. Similarly, racemase efficiently catalyzing racemization using, as a substrate, an acylamino acid, such as N-acylalanine, N-acylaspartic acid, N-acylleucine or N-acylvaline is not yet known.
An objective of the present invention is to provide a method for racemizing N-acylamino acids using a racemase exhibiting racemization activity on a wide variety of substrates and a method for producing optically active amino acids using the racemization method.
In order to achieve these objectives, the present inventors searched for racemases effectively catalyzing a wide variety of substrates. Then, the present inventors have found that the NAAR derived from Sebekia benihana, which was previously reported by the inventors, has the substrate specificity that is industrially available for racemization of N-acylamino acids, thereby completing the present invention. Specifically, the present invention relates to a method for racemizing N-acylamino acid using NAAR having particular substrate specificity and a method for producing optically active amino acids using the racemization method. More specifically, it relates to:
[1] A method for racemizing an N-acylamino acid, the method comprising contacting an N-acylamino acid racemase or a processed product thereof with an optically active N-acylamino acid to racemize the N-acylamino acid, wherein the racemase comprises a polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2;
(b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, inserted, and/or added, and having activity of an N-acylamino acid racemase having activity of an N-acylamino acid racemase having enzymatic properties of (1) and (2) below; and
(c) a polypeptide encoded by a polynucleotide hybridizing to a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and having activity of an N-acylamino acid racemase having enzymatic properties of (1) and (2) below;
(1) action: the racemase racemizes N-acylamino acids and
(2) substrate specificity: the racemase has relative activity of at least 50 or higher for each of N-acylalanine, N-acylaspartic acid, N-acylleucine, N-acylvaline, and N-acyltryptophan among N-acylamino acids when the activity for N-acylmethionine is taken as 100.
[2] A method for racemizing an N-acylamino acid, the method comprising contacting a microorganism producing a racemase or a processed product of the microorganism with an optically active N-acylamino acid to racemize the N-acylamino acid, wherein the microorganism is a transformant expressing a polypeptide encoded by a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the coding region of the nucleotide sequence of SEQ ID NO:1;
(b) a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
(c) a polynucleotide hybridizing to a DNA comprising the nucleotide sequence of SEQ ID NO:1 under stringent conditions, wherein the polynucleotide encodes a polypeptide having activity of an N-acylamino acid racemase having enzymatic properties of (1) and (2) below; and
(d) a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 in which one or more amino acids are substituted, deleted, inserted, and/or added, and having activity of an N-acylamino acid racemase having enzymatic properties of (1) and (2) below;
(1) action: the racemase racemizes N-acylamino acids and
(2) substrate specificity: the racemase has relative activity of at least 50 or higher for each of N-acylalanine, N-acylaspartic acid, N-acylleucine, N-acylvaline, and N-acyltryptophan among N-acylamino acids when the activity for N-acylmethionine is taken as 100.
[3] The method according to [1] or [2], wherein the N-acylamino acid is at least one N-acylamino acid selected from the group consisting of N-acylalanine, N-acylaspartic acid, N-acylleucine, N-acylvaline, and N-acyltryptophan.
[4] A method for producing a D- or L-amino acid, the method comprising racemizing an N-acyl-DL-amino acid by the method according to [1] or [2] in the presence of a D- or L-aminoacylase.